This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lucas, S.
Right arrow Articles by Erauso, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lucas, S.
Right arrow Articles by Erauso, G.
Agricola
Right arrow Articles by Lucas, S.
Right arrow Articles by Erauso, G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, November 2002, p. 5528-5536, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5528-5536.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

Soizick Lucas,1 Laurent Toffin,1 Yvan Zivanovic,2 Daniel Charlier,3 Hélène Moussard,1 Patrick Forterre,2 Daniel Prieur,1 and Gaël Erauso1*

LEMAR, UMR CNRS 6539, IUEM, Université de Bretagne Occidentale, Technopôle Brest-Iroise, 29280 Plouzané,1 Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France,2 Vrije Universiteit Brussel, Erfelijkheidsleer en Microbiologie, B-1070 Brussels, Belgium3

Received 1 March 2002/ Accepted 22 August 2002

Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per µg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

* Corresponding author. Mailing address: LEMAR, UMR CNRS 6539, IUEM, Université de Bretagne Occidentale, Technopôle Brest-Iroise, Place Copernic, 29280 Plouzané, France. Phone: 33 2 98 49 87 50. Fax: 33 2 98 49 87 05. E-mail: gael.erauso{at}univ-brest.fr.

Applied and Environmental Microbiology, November 2002, p. 5528-5536, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5528-5536.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Santangelo, T. J., Cubonova, L., Reeve, J. N. (2008). Shuttle Vector Expression in Thermococcus kodakaraensis: Contributions of cis Elements to Protein Synthesis in a Hyperthermophilic Archaeon. Appl. Environ. Microbiol. 74: 3099-3104 [Abstract] [Full Text]  
  • Matsumi, R., Manabe, K., Fukui, T., Atomi, H., Imanaka, T. (2007). Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance. J. Bacteriol. 189: 2683-2691 [Abstract] [Full Text]  
  • Sato, T., Fukui, T., Atomi, H., Imanaka, T. (2005). Improved and Versatile Transformation System Allowing Multiple Genetic Manipulations of the Hyperthermophilic Archaeon Thermococcus kodakaraensis. Appl. Environ. Microbiol. 71: 3889-3899 [Abstract] [Full Text]  
  • Yano, T., Sanders, C., Catalano, J., Daldal, F. (2005). sacB-5-Fluoroorotic Acid-pyrE-Based Bidirectional Selection for Integration of Unmarked Alleles into the Chromosome of Rhodobacter capsulatus. Appl. Environ. Microbiol. 71: 3014-3024 [Abstract] [Full Text]  
  • Fukui, T., Atomi, H., Kanai, T., Matsumi, R., Fujiwara, S., Imanaka, T. (2005). Complete genome sequence of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 and comparison with Pyrococcus genomes. Genome Res 15: 352-363 [Abstract] [Full Text]  
  • Galvao, T. C., de Lorenzo, V. (2005). Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a {Delta}betCDE Pseudomonas putida Strain. Appl. Environ. Microbiol. 71: 883-892 [Abstract] [Full Text]  
  • Toffin, L., Bidault, A., Pignet, P., Tindall, B. J., Slobodkin, A., Kato, C., Prieur, D. (2004). Shewanella profunda sp. nov., isolated from deep marine sediment of the Nankai Trough. Int. J. Syst. Evol. Microbiol. 54: 1943-1949 [Abstract] [Full Text]  
  • Lepage, E., Marguet, E., Geslin, C., Matte-Tailliez, O., Zillig, W., Forterre, P., Tailliez, P. (2004). Molecular Diversity of New Thermococcales Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis. Appl. Environ. Microbiol. 70: 1277-1286 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»